Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.     

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Maternal protein homeostasis and milk protein synthesis during feeding and fasting in humans

Little is known about amino acid (AA) and protein metabolism in lactating women. We hypothesized: 1) AA sources other than the plasma acid pool provide substrate for milk protein synthesis in humans and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in the lactating women. To test these hypotheses, six healthy exclusively breast-feeding women [27 +/- 3 yr; body mass index (BMI) 26 +/- 2 kg/m2] between 6 wk and 3 mo postpartum and six healthy nonlactating women (28 +/- 2 yr; BMI 22 +/- 1 kg/m2) were studied two times, in random order, during 22 h fasting or 10 h of continuous feeding with a mixed nutrient drink. Protein metabolism was determined using [1-13C]leucine and [15N2]urea. In both the fed and fasted states, a significant portion of milk protein (20 +/- 5 and 31 +/- 6%, respectively) was derived from sources other than the plasma free AA pool. A 70% higher (P < 0.02) FSR of albumin was observed in lactating women during feeding, suggesting that albumin is a likely source of AA for milk protein synthesis. We conclude that plasma free AA contribute only 70-80% of the substrate for milk protein synthesis in humans and that albumin may be a significant source of amino acids for the remainder.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

First Administration of the Fc-Attenuated Anti-β Amyloid Antibody GSK933776 to Patients with Mild Alzheimer’s Disease: A Randomized,Placebo-Controlled Study

Objective

To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-β amyloid (Aβ) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer’s disease (AD) or mild cognitive impairment (MCI).

Methods

This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001–6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1–6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436).

Results

There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or –hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t_1/2) was 10–15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aβ42 and Aβ increased whereas plasma levels of free Aβ decreased dose dependently; no changes were observed for placebo. For total Aβ42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aβ the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aβ showed increases from baseline to week 12 for Aβ X–38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aβ X–42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses.

Conclusion

In this FTIH study the Fc-inactivated anti-Aβ mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI.

Trial Registration

ClinicalTrials.gov NCT00459550     

Maturation of Oral Microbiota in Children with or without Dental Caries

Background

The aim of this longitudinal study was to evaluate the oral microbiota in children from age 3 months to 3 years, and to determine the association of the presence of caries at 3 years of age.

Methods and findings

Oral biofilms and saliva were sampled from children at 3 months (n = 207) and 3 years (n = 155) of age, and dental caries was scored at 3 years of age. Oral microbiota was assessed by culturing of total lactobacilli and mutans streptococci, PCR detection of Streptococcus mutans and Streptococcus sobrinus, 454 pyrosequencing and HOMIM (Human Oral Microbe Identification Microarray) microarray detection of more then 300 species/ phylotypes. Species richness and taxa diversity significantly increased from 3 months to 3 years. Three bacterial genera, present in all the 3-month-old infants, persisted at 3 years of age, whereas three other genera had disappeared by this age. A large number of new taxa were also observed in the 3-year-olds. The microbiota at 3 months of age, except for lactobacilli, was unrelated to caries development at a later age. In contrast, several taxa in the oral biofilms of the 3-year-olds were linked with the presence or absence of caries. The main species/phylotypes associated with caries in 3-year-olds belonged to the Actinobaculum, Atopobium, Aggregatibacter, and Streptococcus genera, whereas those influencing the absence of caries belonged to the Actinomyces, Bergeyella, Campylobacter, Granulicatella, Kingella, Leptotrichia, and Streptococcus genera.

Conclusions

Thus, during the first years of life, species richness and taxa diversity in the mouth increase significantly. Besides the more prevalent colonization of lactobacilli, the composition of the overall microbiota at 3 months of age was unrelated to caries development at a later age. Several taxa within the oral biofilms of the 3-year-olds could be linked to the presence or absence of caries.     

Water transfer via ectomycorrhizal fungal hyphae to conifer seedlings

Little is known about water transfer via mycorrhizal hyphae to plants, despite its potential importance in seedling establishment and plant community development, especially in arid environments. Therefore, this process was investigated in the study reported in this paper in laboratory-based tripartite mesocosms containing the shrub Arctostaphylos viscida (manzanita) and young seedlings of sugar pine (Pinus lambertiana) and Douglas-fir (Pseudotsuga menziesii). The objectives were to determine whether water could be transported through mycorrhizal symbionts shared by establishing conifers and A. viscida and to compare the results obtained using two tracers: the stable isotope deuterium and the dye lucifer yellow carbohydrazide. Water containing the tracers was added to the central compartment containing single manzanita shrubs. The fungal hyphae were then collected as well as plant roots from coniferous seedlings in the other two compartments to determine whether water was transferred via fungal hyphae. In addition, the length of the hyphae and degree of mycorrhizal colonisation were determined. Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) analysis was used to identify the fungal species involved in dye (water) transfer. Results of the stable isotope analysis showed that water is transferred via mycorrhizal hyphae, but isotopically labelled water was only detected in Douglas-fir roots, not in sugar pine roots. In contrast, the fluorescent dye was transported via mycorrhizal hyphae to both Douglas-fir and sugar pine seedlings. Only 1 of 15 fungal morphotypes (identified as Atheliaceae) growing in the mesocosms transferred the dye. Differences were detected in the water transfer patterns indicated by the deuterium and fluorescent dye tracers, suggesting that the two labels are transported by different mechanisms in the same hyphae and/or that different fungal taxa transfer them via different routes to host plants. We conclude that both tracers can provide information on resource transfer between fungi and plants, but we cannot be sure that the dye transfer data provide accurate indications of water transfer rates and patterns. The isotopic tracer provides more direct indications of water movement and is therefore more suitable than the dye for studying water relations of plants and their associated mycorrhizal fungi.     

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.